JPS6120544B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to mercaptoalkylacyl amino acid derivatives, and more particularly to novel amino acid compounds and salts thereof useful as angiotensin converting enzyme inhibitors. By administering the angiotensin converting enzyme inhibitor selected from mercaptoalkylacyl amino acids, derivatives thereof, or salts thereof according to the present invention to a hypertensive mammal, angiotensin-related hypertension can be reduced or alleviated. Angiotensin is a substance that has a strong vasoconstrictor effect, and this substance is known to be related to the main causative agent in the pathogenesis of vascular hypertension. Angiotensin is formed from angiotensin by the action of angiotensin converting enzyme. Angiotensin is a biologically inactive decapeptide produced by the cleavage of angiotensinogen (blood protein) by the action of the enzyme renin (Oparil et al.: New England Journal of Medicine). (New
England J. of Med.) Vol. 291, pp. 389-457 (1974)
). Also, angiotensinogen and renin are each themselves biologically inert substances. In addition, angiotensin-converting enzyme is responsible for inactivating bradykinin, which is known to be involved in the regulation of brain function as a substance with vasodilatory effects (Erdos: Circulation Research). Research）
(See Vol. 36, p. 247 (1975)). Since angiotensin is a substance that only contributes to its conversion to angiotensin, a substance that can suppress angiotensin converting enzyme has the effect of suppressing its blood pressure increasing effect by suppressing the conversion of angiotensin to angiotensin. It can be said that. Such inhibitors can be used therapeutically in the treatment of various types of vascular malignant hypertension and other hypertension caused by angiotensin (Gavras et al.: New England Journal of Medicine). New England J.of Med.
) Vol. 291, p. 817 (1974)). According to Oparil et al., angiotensin is a substance that plays a major role in maintaining dynamic equilibrium in the circulatory system of sodium-deficient animals, but in normal animals that normally consume salt, angiotensin is It is said that it is not an acutely necessary substance to maintain normal blood pressure. In a variety of conditions that stimulate the renin-angiotensin system, acute administration of angiotensin-converting enzyme inhibitors, or angiotensin blockers, can lower blood pressure (which results in increased plasma renin action). Certain mercaptoacylamino acids have previously been described in the literature. For example, mercaptopropionylglycine derivatives enhance liver function and are useful as antidotes to toxins such as mercury and organoarsenic compounds (U.S. Pat. No. 3,246,025 (date of April 12, 1966) and German (See Patent Publication No. 2349707). N-(α-mercaptoacyl) amino acids have been shown to be useful for preventing or treating metabolic disorders such as autotoxicity caused by heavy metal damage, radiation damage, diabetes or hepatitis (U.S. Pat. No.
No. 3897480 (patent date: July 29, 1975). It is also disclosed in US Pat. No. 3,857,951 (Dated December 31, 1974) that 2-mercaptopropionylglycine and its alkali metal salts can be used to treat respiratory diseases. 2-Finding that mercaptopropionylglycine is a hepatoprotective agent and lowers blood pressure when intravenously injected into anesthetized normotensive rats (Schulze: Arzneim.For-
Schwarts: Methods in Pharmacology, Vol.
Page 125 (1971) and Berger:
Selected Pharmacological Testing Methods, Vol. 3, pp. 171 and 194 (1968)), but on the other hand, it has been reported that this compound does not show any significant effect on blood pressure (Fujimura et al., Japanese Pharmacological Journal (English)). ) Vol. 60, pp. 278-292 (1964)) have also been reported. Additionally, it has been reported that α-mercaptopropionylglycine lowers the blood renin concentration in rats with normal blood pressure and increases angiotensinogen through this feedback mechanism (RiPa: Proc.Int.Symp.
See Thiola, Osaka, Japan (1970) pp. 226-230). The inventors have discovered that certain mercaptoacyl amino acids are useful as angiotensin-converting enzyme inhibitors and can reduce or alleviate angiotensin-related hypertension when administered to hypertensive mammals. The novel compound according to the present invention is represented by the following formula [I], and angiotensin-related hypertension can be alleviated or alleviated by administering the compound to a hypertensive mammal. [Wherein R ₁ is hydrogen, lower alkyl, phenyl (lower) alkyl or guanidino (lower) alkyl; R ₂ is hydrogen or lower alkyl; R ₃ is hydrogen, lower alkyl or phenyl (lower) alkyl; R ₅ is hydrogen , lower alkanoyl or benzoyl; asterisk (*) represents an asymmetric center] Compounds of the present invention ( ) are angiotensin-converting enzyme inhibitors for reducing or alleviating renin-angiotensin-related hypertension in mammals such as rats, dogs, etc. renin-angiotensin-related hypertension includes, for example, vascular hypertension and malignant hypertension.As is clear from the above, the present invention Compound〔〕
A method of reducing or alleviating renin-angiotensin associated hypertension is achieved by administering any of the compounds or mixtures thereof. The method comprises administering to a mammal with renin-angiotensin related hypertension an effective amount of an angiotensin-converting enzyme inhibitor selected from compounds [ ]. The groups represented by each symbol in the above formula have the following meanings. Lower alkyl includes straight chain or branched hydrocarbon groups having not more than 7 carbon atoms (eg, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, inpentyl, hexyl, heptyl, etc.). Phenyl (lower) alkyl includes the same lower alkyls bonded to phenyl. Among these, benzyl and phenethyl are particularly preferred, and benzyl is most preferred. Lower alkanoyl includes acyl residues of lower fatty acids (eg, acetyl, propionyl, butyryl, etc.). Among these, C ₂ to C ₄ are preferred. The compounds of the present invention [ ] are derived from amino acids. It therefore contains amino acids in its structure.
Among these amino acids, alanine, leucine, phenylalanine, arginine, and sarcosine are preferred. R ₅ is preferably hydrogen. Among the compounds of the present invention [], N〓-(3-mercapto-2-methylpropanoyl)-L-arginine, N〓-(3-mercaptopropanoyl)-L
-Arginine, N-(3-mercaptopropanoyl)-L-phenylalanine and N-(3-mercaptopropanoyl-L-leucine are preferred compounds having the highest pharmacological activity, and are particularly preferred embodiments of the present invention. The compound of the present invention [ ] forms salts with various inorganic bases and organic bases, and these salts are also included within the scope of the compound of the present invention. These salts include ammonium acid, alkali metal salts ( salts with organic bases (e.g. dicyclohexylamine salts, benzathine salts, hydrabamine salts, N-methyl-D-glucamine salts). The compound of the present invention [] has two asymmetric carbon atoms when R ₁ and R ₃ are groups other than hydrogen.
In the formula [], these asymmetric carbon atoms are indicated with an asterisk (*). Therefore, the compounds of the present invention [] exist as diastereomers or racemic mixtures, and all such compounds are also included within the scope of the compounds of the present invention. In order to obtain a significant degree of biological activity, the compound of the present invention [ ] should be in the L-configuration with respect to the asymmetric carbon atom to which its R ₁ group is attached;
Regarding such stereoisomerism, nothing is disclosed in conventional patent applications related to mercaptoacylamino acids. The compound of the present invention [ ] is preferably an L-type compound. The angiotensin-converting enzyme inhibitory activity of the compound of the present invention [] was determined in vitro by the method of Cushman and Cheung using smooth muscle samples excised from rabbits and angiotensin-converting enzyme isolated from rabbit lungs. (Biochem.Pharmacol. for the method of Kutsyuman et al. and EO′Keefe et al. for the smooth muscle sample testing method:
(See Federation Proc. Vol. 31, p. 511 (1972)).
Through such tests, it was found that the compound of the present invention strongly suppresses the effect of shortening the chain length of angiotensin and has the effect of shortening the chain length of bradykinin. In the present invention, by administering an angiotensin-converting enzyme inhibitor or one of its physiologically acceptable salts or a mixture thereof to a mammal suffering from angiotensin-induced hypertension, this is mediated by the renin→angiotensin→angiotensin conversion system. Its symptoms can be reduced or alleviated. In order to lower blood pressure that increases due to factors related to angiotensin, the compounds of the present invention are
1000mg/Kg/day, preferably about 10-500mg/Kg/
This should be done once a day, especially at a dosage of 30-300mg/Kg/day.
It is appropriate to administer once a day, preferably in 2 to 4 divided doses. For standard animal experimental procedures,
Engel (SLEngel), Shehua (TR
Schaefer, MHWaugh and R.Rubin: Pro.Soc.Exp.Biol.Med.Vol. 143, 483.
(1973), which provides useful experimental guidance. The inhibitor of the present invention is preferably administered orally, but subcutaneously,
Administration can be by parenteral methods such as intramuscular, intravenous or intraperitoneal administration. The starting material for producing the target compound [] can be obtained by the following method: Formula: R ₆ COSH [] [wherein R ₆ represents lower alkyl or phenyl]. ] Thioacid and formula: [In the formula, R ₃ has the same meaning as above. ] By reacting acrylic acid represented by the formula: [In the formula, R ₃ and R ₆ have the same meanings as above. ] The starting material shown can be obtained. The compound of the present invention [] can be produced by the following method using the above starting material []. That is, the starting material [] and the formula: [In the formula, R ₁ and R ₂ have the same meanings as above. ] By coupling the amino acids shown by the formula: [In the formula, R ₁ , R ₂ , R ₃ and R ₆ have the same meanings as above. ] The compound of the present invention [ ] in which compound R ₅ is lower alkanoyl or benzoyl can be obtained. Then, by ammonolysis treatment of this product [], the formula: [In the formula, R ₁ , R ₂ and R ₃ have the same meanings as above. ] The compound represented by (the compound of the present invention [] in which R ₅ is hydrogen) can be obtained. The compound of the present invention [] can be produced by another method described below. Formula: [Wherein, X represents halogen (preferably chloro or bromo). R ₃ has the same meaning as above] is activated by a conventional method to produce its active derivatives (including mixed acid anhydrides, symmetric anhydrides, acid halides, active esters, etc.). The haloalkanoic acid or its active derivative and the amino acid [ ] are coupled to form a compound of the formula: [In the formula, R ₁ , R ₂ , R ₃ and X have the same meanings as above] A compound represented by the following is prepared. Then, by treating this compound [] with the thioic acid [] to replace its anion, the formula: [In the formula, R ₁ , R ₂ , R ₃ and R ₆ have the same meanings as above] A compound of the present invention [] in which R ₅ is lower alkanoyl or benzoyl can be obtained. Then, this compound [] '] can be converted into the corresponding mercapto compound in the same manner as above to obtain the compound of the present invention in which R ₅ is hydrogen. The compound of the present invention [] can be formulated into a drug such as a tablet, capsule, or elixir. For parenteral administration, the compounds of the present invention can be used as sterile aqueous solutions or suspensions.The present invention Compound [] or a physiologically acceptable salt thereof or a mixture thereof from about 10 to
a physiologically acceptable medium, carrier, in an amount of 500 mg;
They can be formulated into unit dosage forms compatible with pharmacological practice by incorporating excipients, binders, preservatives, stabilizers, flavoring agents, and the like. The amount of active ingredient in a unit drug should be such that the active ingredient is contained in the dosage form in proportions within the ranges mentioned above. Examples of adjuvants that can be incorporated into tablets, capsules, etc. are: binders such as gum tragacanth, gum arabic, cornstarch, gelatin; excipients such as dicalcium phosphate; cornstarch, Disintegrants such as potato starch, alginic acid, etc.; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose or saccharin; flavoring agents such as peppermint, wintergreen oil or thierry flavor. When the dosage form is a capsule, it can contain, in addition to the adjuvants enumerated above, a liquid carrier such as a fatty oil. In addition, various substances can be used as coating agents or adjuvants for other purposes to enhance the pharmaceutical quality of the drug. For example, shellac, sucrose and the like can be used as coating agents for the tablets. A syrup or elixir may contain sucrose as the active ingredient and a sweetening agent, methyl and propylparabens as preservatives, and thiery flavor or orange flavor as a coloring agent and flavoring agent. The active substances of the invention are dissolved or suspended in conventional carriers such as sterile water for injection, natural vegetable oils (e.g. sesame oil, coconut oil, peanut oil, cottonseed oil, etc.) or fatty synthetic carriers (e.g. ethyl oleate). Sterile pharmaceutical compositions for injection can be formulated according to conventional pharmaceutical practice. Buffers, preservatives, antioxidants, etc. can be added to the various drugs mentioned above, if necessary. Next, a method for producing a preferred compound [] of the present invention will be described in detail with reference to Examples. Example 1 N-(3-benzoylthiopropanoyl)-L
-Production of alanine:- Dissolve 4.45 g of L-alanine in 50 ml of 1N aqueous sodium hydroxide solution, and cool this solution while stirring in an ice bath. 27ml of 2N sodium hydroxide and 3
- 8.5 g of bromopropionyl chloride are added in this order, the ice bath is removed and the mixture is stirred at room temperature for 3.5 hours. Thiobenzoic acid 7.5g, potassium carbonate 4.8g
and 50 ml of water are added and the resulting mixture is stirred at room temperature overnight. After acidifying with concentrated hydrochloric acid, the aqueous solution is extracted with ethyl acetate, the organic layer is washed with water, dried, and concentrated to dryness. 14.9 g of the residue was crystallized from ether to obtain 7.1 g of N-(3-benzoylthiopropanoyl)-L-alanine. Melting point 99-100
℃. Example 2 Production of N-(3-mercaptopropanoyl)-L-alanine:- N-(3-benzoylthiopropanoyl)-L
-Dissolve 4.2 g of alanine in a mixture of 7.5 ml of water and 6 ml of concentrated ammonium hydroxide. After 1 hour, the mixture is diluted with water, filtered and the filtrate is extracted with ethyl acetate. The aqueous layer is made acidic with concentrated hydrochloric acid and extracted with ethyl acetate. The organic layer is washed with water, dried, and then concentrated to dryness under reduced pressure. The residue was crystallized from ethyl acetate-hexane to give N-(3-mercaptopropanoyl)-L.
- Obtained 1.87g of alanine. Melting point 79-81℃. Example 3 N-(3-benzoylthiopropanoyl)-L
-Production of leucine:- Using 6.55 g of L-leucine in place of L-alanine in the treatment of Example 1, the same treatment was performed to obtain crude N-(3-benzoylthiopropanoyl)-L-
Obtain 16.7 g of leucine. Add this substance to ethyl acetate
Dissolve in a mixture of 200 ml and dicyclohexylamine, and filter and dry the crystalline salt formed. yield
19.5g, melting point 178-180℃. This dicyclohexylammonium salt is treated with a mixture of 200 ml of ethyl acetate and 50 ml of 10% aqueous potassium hydrogen sulfate solution. The organic layer was dried over magnesium sulfate, concentrated to dryness under reduced pressure, and the residue was crystallized from ethyl acetate-hexane to obtain 8.8 g of pure N-(3-benzoylthiopropanoyl)-L-leucine. Melting point 99~
101℃. Example 4 Production of N-(3-mercaptopropanoyl)-L-leucine:- In place of N-(3-benzoylthiopropanoyl)-L-alanine in the treatment of Example 2, N-
6.46 g of (3-benzoylthiopropanoyl)-L-leucine was treated in the same manner to obtain 2.75 g of N-(3-mercaptopropanoyl)-L-leucine. Melting point 131-132℃. The resulting material was recrystallized from acetonitrile. Example 5 N-(3-benzoylthiopropanoyl)-L
-Production of phenylalanine:- Using 8.25 g of L-phenylalanine in place of L-alanine in the treatment of Example 1, the same treatment was performed to produce N-(3-benzoylthiopropanoyl).
-18.8g of L-phenylalanine was obtained. This material was crystallized from acetonitrile to give 11.1 g of product. Melting point 123-124°C Example 6 Production of N-(3-mercaptopropanoyl)-L-phenylalanine:- N-(3-benzoylthiopropanoyl)-L
-Dissolve 1.78 g of phenylalanine in a mixture of 20 ml of water and 5.5 ml of 1N sodium hydroxide. Add 20 ml of concentrated ammonium hydroxide and then 20 ml of water to this solution. After 3 hours, the mixture was extracted with ethyl acetate and
Acidify with concentrated hydrochloric acid and re-extract with ethyl acetate. This second ethyl acetate extract is washed with water, dried over magnesium sulfate, and then concentrated to dryness under reduced pressure. The residue was chromatographed on a silica gel column with a benzene-acetic acid mixture to give 0.47 g of N-(3-mercaptopropanoyl)-L-phenylalanine.
I got it. Melting point 106-107℃. Example 7 N〓-(3-benzoylthiopropanoyl)-
Production of L-arginine: - Dissolve 8.7 g of L-arginine in 50 ml of 1N aqueous sodium hydroxide solution, and cool this solution while stirring in an ice bath. 25ml of 2N sodium hydroxide and 3
-8.5 g of bromopropionyl chloride are added in this order, the mixture is removed from the ice bath and stirred at room temperature for 2 hours. 7.5g thiobenzoic acid, potassium carbonate
A mixture of 2.4 g and 10 ml of water is added and the resulting mixture is stirred at room temperature overnight. Ion exchange resin (polystyrene sulfonic acid resin: Dowex 50 (Mikes): Laboratory Handbook of Chromatographic
Methods), Juan Nostrand (Van
Nostrand (1961), p. 256)) is added, and the suspension is passed through a 300 ml column of the same resin. Wash with water to remove acidic substances, and elute N-(3-benzoylthiopropanoyl)-L-arginine with pyridine-acetic acid-water buffer (PH6.5). The fractions containing this material were combined and concentrated to dryness.
Treatment of the residue with ether gave 7 g of product.
Melting point 345℃ (decomposition). Example 8 N〓-(3-mercaptopropanoyl)-L-
Preparation of arginine: 1 g of N-(3-benzoylthiopropanoyl)-L-arginine is dissolved in a mixture of 5 ml of water and 5 ml of concentrated ammonia while stirring vigorously. 1
After some time, the solution is extracted with ethyl acetate and concentrated to dryness under reduced pressure. The residue was filtered onto a DEAE Sephadex (anion exchange resin derived from dextran (see Mikes reference, p. 328)) 85 ml column.
Chromatograph with 0.005M ammonium bicarbonate buffer. Fractions containing N-(3-mercaptopropanoyl)-L-arginine (detected by thiol positivity and Sakaguchi reaction) were combined;
The ammonium bicarbonate was removed by lyophilization to obtain 200 mg of product. Melting point: 230℃ (starts to decompose at 200℃). Example 9 Production of N-(3-benzoylthiopropanoyl)sarcosine: - Using 4.45 g of sarcosine in place of L-alanine in the treatment of Example 1, N-
(3-benzoylthiopropanoyl)sarcosine
7.9g was obtained. Melting point 139-140℃. Example 10 Preparation of N-(3-mercaptopropanoyl)sarcosine: - Substituting N-(3-benzoylthiopropanoyl)-L-alanine in the treatment of Example 2
-(3-benzoylthiopropanoyl)sarcosine (2.8g) was treated in the same manner to obtain 1.65g of crude N-(3-mercaptopropanoyl)sarcosine. This material is converted to its dicyclohexylammonium salt. Yield 2.7g, melting point 157-158°C.
The purified salt was converted to the free acid by partitioning it between ethyl acetate and 10% potassium hydrogen sulfate. Example 11 Production of N〓(3-acetylthio-2-methylpropanoyl)-L-arginine:- 2.61g of L-arginine and 3.2g of sodium carbonate
and 30 ml of water and cool the solution in an ice bath. 2.7 g of 3-acetylthio-2-methylpropanoyl chloride are added and the mixture is stirred at room temperature for 1.5 hours. Add 50 ml of ion exchange resin (AG50W) and run the suspension through an 80 ml column of the same resin. After washing with water, add N〓- to pyridine-acetate buffer (PH6.5).
(3-acetylthio-2-methylpropanoyl)
-L-arginine was eluted, the solvent was removed under reduced pressure, the residue was dissolved in methanol, and the product was precipitated with ether to give N〓-(3-acetylthio-2
-Methylpropanoyl)-L-arginine 3.86g
I got it. Melting point 133℃. Example 12 Production of N-(3-mercapto-2-methylpropanoyl)-L-arginine: - 1 g of N-(3-acetylthio-2-methylpropanoyl)-L-arginine was mixed with 5 ml of water and concentrated ammonia. Dissolve in 5 ml of mixture. After standing at room temperature for 1 hour, the solution was diluted with 3 ml under reduced pressure (without heating).
Concentrate and add ion exchange resin (AG-50W) until the pH is approximately 4. This suspension is passed through a similar resin column, and N-(3-mercapto-2-methylpropanoyl)-L-arginine is extracted with pyridine-acetate buffer (PH6.5). The solvent was removed under reduced pressure and the residue was lyophilized to yield 0.86 g of product.
I got it. Melting point 100℃. Reference Example 1 Production of 3-acetylthio-2-benzylpropanoyl chloride: 8.1 g of 2-benzyl acrylic acid and 5.3 g of thiol acetic acid are mixed and heated on a steam bath for 1 hour. After cooling to room temperature, 9.75 g of thionyl chloride are added and the mixture is left at room temperature overnight. Excess thionyl chloride was removed under reduced pressure, and the residue was distilled to obtain 3-acetylthio-2-benzylpropanoyl chloride. Boiling point (0.05mm) 120-122℃. Example 13 Production of N-(3-acetylthio-2-benzylpropannoyl)-L-arginine: 3-acetylthio-2 in the treatment of Example 20
- Using 3-acetylthio-2-benzylpropanoyl chloride instead of methylpropanoyl chloride, the same treatment was performed to obtain N〓-(3-acetylthio-
2-Benzylpropanoyl)-L-arginine was obtained. Melting point 253-295℃. Example 14 Production of N-(3-mercapto-2-benzylpropanoyl)-L-arginine: - Production of N-(3-acetylthio-2-methylpropanoyl)-L-arginine in the treatment of Example 21 Instead, N-(3-acetylthio-2-benzylpropanoyl)-L-arginine was used and treated in the same manner to obtain N-(3-mercapto-2-benzylpropanoyl)-L-arginine. Melting point 135℃. Example 15 Production of 1000 tablets containing 100 mg of N-(3-mercaptopropanoyl)-L-phenylalanine per tablet: Ingredients: N-(3-mercaptopropanoyl)-
L-phenylalanine 100g, cornstarch 50g
g, 7.5 g gelatin, 25 g Avicel (microcrystalline cellulose) and 2.5 g magnesium stearate. The above N-(3-mercaptopropanoyl)-L
- Mix phenylalanine and cornstarch with aqueous gelatin solution. This mixture is dried and ground to obtain a fine powder. Avicel and then magnesium stearate are added and mixed with the granulating agent to the above powder to produce granules. This was compressed using a compression tablet machine to produce 1000 tablets containing 100 mg of active ingredient per tablet. Example 16 Production of 1000 tablets containing 200 mg of N-2-mercaptopropanoylglycine per tablet:
- Ingredients: 200g N-2-mercaptopropanoylglycine, 100g lactose, 150g Avicel, 50g cornstarch and 5g magnesium stearate. Mix N-2-mercaptopropanoylglycine, lactose and Avicel, then mix with cornstarch and add magnesium stearate. The dry mixture was compressed in a compression tablet machine to produce 1000 505 mg tablets containing 200 mg of active ingredient per tablet.
Manufacture pieces. The tablets are coated with a solution of Methocel E15 (methylcellulose) containing a lake containing Yellow #6 as a colorant. Example 17 Production of solution for injection: - Ingredients: N - (3-mercaptopropanoyl) -
L-phenylalanine 500g, methylparaben 5
g, propylparaben 1g, sodium chloride 25g
and water for injections (total amount 5000 including water for injections)
ml). Dissolve the above active ingredients, preservative and sodium chloride in 3000 ml of water for injection, then reduce the volume to
Make it 5000ml. This solution is filtered through a sterile filter, filled aseptically into a small bottle that has been sterilized in advance, and the bottle is sealed with a rubber stopper that has been sterilized in advance.
1000 small bottles filled with 5 ml of solution containing 100 mg of active ingredient per ml of solution for injection were obtained. Example 18 Since the reaction of converting angiotensin to angiotensin by angiotensin converting enzyme is probably the most important reaction in the pathology of hypertension, in order to test the activity of the isolated enzyme, the activity of converting angiotensin to angiotensin was measured. However, its activity can be tested more accurately and conveniently by measuring the rate of cleavage of the simpler peptide substrate hipryl-L-histidyl-L-leucine. To determine the I ₅₀ value (the amount of compound required to inhibit angiotensin-converting enzyme by 50%, expressed as a concentration in μg/ml), each component was dissolved in potassium phosphate buffer in a total volume of 0.25 ml. (PH8.3) 100mM, sodium chloride
Various concentrations of active compound are added to 13 x 100 mm test tubes containing each component to give a final concentration of 300 mM hipryl-L-histidyl-L-leucine, 5 mM rabbit lung angiotensin converting enzyme, and 5 milliunits of rabbit lung angiotensin converting enzyme. At the same time, a control sample containing no inhibitor (100% enzyme activity) and a control sample that was acidified before enzyme addition (0% enzyme activity) were prepared. The time when the enzyme component is added is defined as the start point of the enzyme reaction,
Each test tube was kept at 37°C for 30 minutes to react, and the reaction was terminated by adding 1N hydrochloric acid. Hypril-L-
Hippuric acid produced by the action of angiotensin converting enzyme on histidyl-L-leucine is extracted with ethyl acetate, evaporated to dryness, redissolved in water, and quantified from its absorption at 228 nm. Calculate the % inhibition of each concentration of compound by comparing to control samples of 0% and 100% enzyme activity. The concentrations of representative compounds of the present invention at which the representative compounds of the present invention can inhibit the angiotensin inhibitory enzyme action by 50% are shown below.

[Table] Example 19 In order to evaluate the inhibitory effect on angiotensin converting enzyme in the ileum excised from guinea pigs, the following test was conducted: Various concentrations of activity were added to Krebs solution (37°C) containing the excised guinea pig ileum. Add the compound and aerate with a mixture of 95% oxygen-5% carbon dioxide. After 2 minutes, angiotensin (25 ng/ml) is added, and the isotonic shortening rate of the peptide molecular chain is measured. The concentration (IC ₅₀ ) at which the compound of the present invention inhibits the shortening effect on the molecular chain of angiotensin by 50% is shown below.

【table】

Claims (1)

[Claims] 1 Formula: Compounds and salts thereof [wherein R ₁ is hydrogen, lower alkyl, phenyl (lower) alkyl or guanidino (lower) alkyl; R ₂ is hydrogen or lower alkyl; R ₃ is hydrogen, lower alkyl or phenyl (lower) ) alkyl; and R ₅ is hydrogen, lower alkanoyl or benzoyl].